Supplementary material
Analysis of hepatic deiodinase 2 mRNA levels in natural fish lake populations exposed
to different levels of putative thyroid disrupters
Sergio Jarquea,b, Carme Boscha, Joan O. Grimalta, Demetrio Raldúaa, Benjamin Piñaa,*
a) Institute of Environmental Assessment and Water Research (IDAEA-CSIC). Jordi
Girona, 18. 08034 Barcelona, Spain
b) Masaryk University, Faculty of Science, RECETOX, Kamenice 5/753, Brno
CZ62500, Czech Republic
*Corresponding Author:
Benjamin Piña
IDAEA-CSIC
Jordi Girona, 18. 08034 Barcelona, Spain
Tel: 34 934006157
e-mail:bpcbmc@cid.csic.es
Analytical procedures.
Hepatic mRNA analysis by RT-qPCR. Liver samples were homogenised in TRIzol
Reagent (Gibco, Paisley, UK) using Eppendorf-fitting, RNase free pestles (Iberlabo,
Madrid, Spain). RNA was extracted in TRIzol as specified by the supplier. Total RNA
concentration was estimated by spectrophotometric absorption at 260 nm in a Nanodrop
Spectrophotometer ND-1000 (NanoDrop Technologies; Delaware, USA) and treated
with DNAse I (F. Hoffmann-La Roche Ltd, Basel, Switzerland) to remove
contaminating genomic DNA. RNA integrity was assessed by electrophoresis in a
Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA). RNA was treated
with DNAseI to remove genomic DNA contamination. Ten µg of DNaseI-digested
RNA were copied to cDNA by reverse transcriptase (Omniscript, Qiagen, Valencia,
CA, USA) and stored at -20ºC. Aliquots corresponding to 40 ng of the original RNA
preparation were used to quantify specific transcripts in a Abi Prism 7000 Sequence
Detection System (Applied Biosystems, Foster City, CA, USA) by the SYBR GREEN
method (Applied Biosystems). Sequences for dio2, cyp1a and ß-Actin primers used in
this work were the following:
ß-Actin Forward: 5'- CTGTCTTCCCCTCCATCGTC-3'
ß-Actin Reverse: 5'- TCTTGCTCTGAGCCTCGTCTC-3'
dio2 Forward: 5'- CGCTCCTTCGAGGTCAGG -3'
dio2 Reverse: 5'-ACCACCCTCTCCTCCAGTGAT-3'
CYP1A Forward: 5'-CACTGACTCCCTCATTGACCAC-3'
CYP1A Reverse: 5'-ACAGATCATTGACAATGCCCAC-3'
The relative amounts of cDNA present in the samples were calculated from the
number of cycles required for amplification reaction to reach fluorescence above the
threshold level in the RT-qPCR reaction (CT values), according to the equation 1:
mRNATG EAct(CTAct )
=
mRNAAct ETG (CTTG )
In which EAct and ETG correspond to Real-Time efficiency for ß-actin and the target
gene, respectively. Efficiency values for dio2, cyp1a and ß-Actin primers on real
samples were calculated using a series of standard dilutions as 102%, 101%, and 106%,
respectively. These values were considered close enough one each other and to 100% to
not to require any correction for efficiency on the calculations of relative mRNA
abundance values. The suitability of ß-Actin as reference gene for these samples has
been tested previously2. Results are expressed as copies of dio2 or cyp1a mRNA per
1000 copies of ß-actin mRNA. A typical experiment calculated CT values as averages of
three replicates.
To confirm the sequence of the amplified PCR products, they were purified
using GFX PCR Purification Kit (Amersham Biosciences, Backinghamshire, UK) and
inserted into the pTZ57R/T plasmid (InsT/Aclone PCR Product Cloning Kit, Fermentas,
Burlington, Canada). DNA sequencing was performed using Applied Biosystems 3730
DNA Analyzer (Applied Biosystems). Amplified sequences were compared to
previously reported sequences of homologous genes from different salmonids using
ClustalW from Bioedit Sequence Alignment Editor (BioEdit v7.0.5, Ibis Therapeutics,
Carlsbad, CA).
PCBs and PBDE analysis.
Muscle samples were ground with activated sodium sulphate until a fine powder was
obtained. Cellulose cartridges were filled with this mixture and TBB and PCB209 were
added as standards. A Soxhlet extraction was done with n-hexane:dichloromethane (4:1)
for 18 h. The extract was cleaned up with sulfuric acid (5 times), concentrated by
vacuum rotatory evaporation (20ºC, 20 Torr) and concentrated to near dryness under a
gentle flow of nitrogen. Finally, the extract was redissolved in 50µl of isooctane. Before
chromatographic analysis, an internal standard of PCB142 was added to correct for
instrument variability. Samples were analyzed for PCBs by GC-ECD (Hewlett-Packard
5890 series II) with a 60 m x 0.25 mm i.d. DB-5 capillary column (J&W Scientific,
Folsom, CA) coated with 5% phenyl/95% methylpolysiloxane (film thickness 0.25 m).
The GC operated in splitless mode, and the oven temperature program started at 90ºC
(held for 2 min), ramped to 150ºC at 15ºC·min-1 and then ramped to 290ºC at 4ºC·min-1
(holding time 20 min). Injector and detector temperatures were 280 and 310ºC,
respectively. Helium and nitrogen were used as carrier (1.5 mL·min-1) and makeup (60
mL·min-1) gases, respectively. PBDE were analyzed by negative ion chemical
ionization mass spectrometry coupled to gas chromatography (GC-MS-NICI). A GC
system from Agilent Technologies 6890A (USA) was coupled to an MS detector
5973N. The system was equipped with a HP-5MS capillary column (60 m x 0.25 mm x
0.25 m film thickness). The oven temperature program was from 110ºC (held for 1 min)
to 180ºC at 8ºC·min-1 (held for 1 min), then to 240ºC at 2ºC·min-1 (held for 5 min) and
finally to 310ºC at 2ºC·min-1 (held for 15 min). Helium was used as a carrier gas (10
psi) and ammonia as ionization gas (1.6·10 Pa). Transfer line and quadrupole
temperatures were 280 and 150ºC, respectively. Quantification was performed at a m/z
value of 79 [Br]- which is the base peak of all PBDEs monitored. Confirmation was
done at m/z values of 81 [Br]-, 161 [HBr2]-, 327, 405, 483, 563 and 643, corresponding
to [M]- or [M-HBr 2]-. Retention time shifts could not be higher than 1 sec.
1
2
Pfaffl, M. W. A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Research 29 (2001).
Quirós, L. et al. Physiological response to persistent organic pollutants in
fish from mountain lakes: Analysis of Cyp1A gene expression in natural
populations of Salmo trutta. Environmental Science & Technology 41, 51545160, doi:10.1021/es070455p (2007).
Table ST1. Physical characeristics of lakes sampled in this study
Region
Lake name
Latitude N
Longitude E
Altitude ASL
(m)
Average
Temperature
(ºC)
Pyrenees
Vidal d'Amunt
42.53281
0.99351
2688
1.0
Pyrenees
Xic de Colomina
42.52149
0.99564
2425
2.7
Pyrenees
Cavallers
42.59257
0.85779
1800
5.5
Pyrenees
Llebreta
42.55083
0.89031
1620
6.2
Tatra Mountains
Veľké Hinçovo
49.17970
20.06060
1946
-0.7
Tatra Mountains
Morskie Oko
49.19780
20.07220
1395
2.4
Table ST2. Chemical and gene expression data for the surveyed fish populations
Unit
Llebreta
Median
n
number of
fish
Sexa
Age
years
Lenght
cm
Weight
g
dio2
Cavallers
Min. - Max.
Median
Xic de Colomina
Min. - Max.
Median
Min. - Max.
Vidal d'Amunt
Median
Min. - Max.
Morskie Oko
Median
Veľké Hinçovo
Min. - Max.
Median
14
14
12
12
13
14
1.93
1.71
2.00
1.75
1.25
1.11
6
33
4 - 9
26 - 36
4
28
3 - 7
15 - 32
6
24
21 - 29
30
6
3 - 17
6
5 - 7
24 - 33
18
13 - 54
16
14 - 18
185 - 355
57
18 - 2400
45
23 - 56
273
30 - 430
mRNA copiesb
0.63
0.03 - 7.62
3.03
0.45 - 40.39
cyp1a
mRNA copiesb
271.69
aHCH
ng/gc
0.40
0.14 - 0.55
0.32
0.25 - 1.18
0.10
0.05 - 1.65
0.21
0.11 - 0.92
0.14
0.06 - 0.48
0.27
0.15 - 0.38
HeCB
ng/g
0.40
0.11 - 0.59
0.48
0.33 - 0.79
0.39
0.17 - 0.73
0.58
0.36 - 1.45
0.12
0.01 - 0.65
0.36
0.29 - 0.48
gHCH
ng/g
0.69
0.43 - 4.83
1.02
0.19 - 2.30
1.48
0.91 - 3.67
2.04
1.64 - 3.45
1.16
0.13 - 2.48
2.54
0.32 - 3.80
PCB28
ng/g
0.24
0.03 - 0.49
0.18
0.09 - 0.45
0.27
0.15 - 0.42
0.24
0.04 - 1.03
0.24
0.07 - 1.17
0.45
0.11 - 1.34
PCB52
ng/g
0.31
0.04 - 0.61
0.08
0.02 - 0.33
1.89
0.54 - 3.50
0.31
0.01 - 0.42
0.27
0.12 - 0.63
0.18
0.02 - 0.85
PCB101
ng/g
0.11
0.03 - 0.16
0.39
0.12 - 0.67
0.24
0.01 - 0.67
0.29
0.06 - 0.64
0.19
0.07 - 12.27
1.11
0.22 - 1.77
PCB118
ng/g
0.47
0.21 - 0.78
0.64
0.40 - 0.91
0.77
0.10 - 1.67
0.62
0.23 - 1.51
0.22
0.09 - 19.99
0.31
0.16 - 36.41
PCB153
ng/g
1.21
0.62 - 2.02
1.44
0.56 - 2.39
2.52
1.00 - 6.31
2.67
1.27 - 12.97
1.75
0.36 - 208.66
5.74
3.58 - 8.23
PCB138
ng/g
1.05
0.63 - 1.63
1.48
0.23 - 1.89
2.25
0.89 - 5.91
2.49
1.35 - 11.96
1.20
0.32 - 147.27
6.97
0.68 - 11.26
PCB180
ng/g
0.84
0.47 - 1.32
0.85
0.51 - 1.47
1.59
0.54 - 4.08
1.13
0.59 - 6.59
1.29
0.28 - 231.17
4.74
2.52 - 6.30
ppDDE
ng/g
4.38
2.00 - 6.50
3.37
2.05 - 5.38
7.70
4.00 - 19.30
9.79 - 35.28
9.61
2.51 - 1273.72
p,p´DDT
ng/g
0.60
0.46 - 0.82
0.71
0.50 - 1.26
1.08
0.76 - 2.15
0.66 - 2.48
0.56
0.22 - 30.25
PBDE 28+33
pg/g
78.54
41.33 - 104.23
100.14
99.00 - 172.67
286.88
158.64 - 515.15
248.19
190.12 - 535.22
76.60
36.47 - 172.74
87.81
3.17 - 144.99
PBDE 47
pg/g
339.85
135.59 - 610.72
333.83
188.68 - 438.69
425.87
369.02 - 531.13
545.57
356.42 - 948.59
122.02
55.59 - 589.35
108.82
71.79 - 253.82
PBDE 100
pg/g
168.45
101.72 - 308.49
134.84
112.83 - 500.76
286.93
137.18 - 332.88
256.60
138.75 - 796.55
42.58
PBDE 99
pg/g
237.82
133.33 - 392.60
328.97
167.99 - 432.98
292.35
224.14 - 394.90
304.97
232.22 - 636.08
136.28
PBDE 154
pg/g
80.16
62.39 - 233.75
201.59
65.21 - 226.32
292.25
248.79 - 334.06
257.11
181.45 - 730.97
35.42
11.92 - 51.62
33.55
21.01 - 39.46
77.42 - 149.79
95.45
62.84 - 107.07
246.69
212.41 - 362.58
243.58
123.54 - 512.42
30.06
12.36 - 39.92
24.00
13.16 - 27.59
PBDE153
pg/g
93.66
a) 1=male, 2= female, averages
b) Referred to 1000 copies of ß-actin mRNA
c) Weigh per gram of net tissue (muscle)
383.72
48.70 - 2907.95
1084.79
3.25 - 104.19
632.88 - 2789.49
280
5 - 9
190 - 560
14.63
105 - 215
5
358
38.21 - 486.33
150
5 - 8
Min. - Max.
6.34
871.07
15.62
1.19
0.95 - 40.67
309.93 - 1414.21
1.48
717.25
0.29 - 12.45
57.51 - 2907.95
16.07 - 76.97
61.16 - 764.32
0.87
1328.69
48.04
1.59
31.42
120.40
0.04 - 2.82
1013.96 - 2203.81
29.56 - 69.11
0.32 - 2.50
3.55 - 37.38
76.92 - 153.78
Supplementary Table ST3. Analysis of the distribution of age and reproductive status' values
among the sampled fish populations
Descriptives
n
Mean
Std.
Deviation
Age
Llebreta
Cavallers
Xic de Colomina
Vidal d'Amunt
Morskie Oko
Veľké Hinçovo
Total
14
14
12
12
13
14
79
6.214285714
4.5
6.25
5.416666667
6.384615385
5.857142857
5.759493671
1.42389344
1.224744871
0.753778361
1.164500153
2.218800785
0.949262293
1.486824097
Reproductive status
Llebreta
Cavallers
Xic de Colomina
Vidal d'Amunt
Morskie Oko
Veľké Hinçovo
Total
14
14
12
12
12
14
78
2.642857143
2.714285714
1.833333333
2.833333333
2.866666667
2.714285714
2.607692308
1.823232246
1.437335753
0.937436867
1.403458931
1.952775802
1.38278267
1.51681221
df
Mean
Square
ANOVA
Sum of
Squares
Age
Reproductive status
F
Sig.
Between Groups
34.61536143
5 6.923072286 3.667120486 0.005137304
Within Groups
137.8150183
73 1.887876963
Total
172.4303797
78
Between Groups
Within Groups
Total
8.946813187
168.2085714
177.1553846
5 1.789362637 0.765918816 0.577376731
72 2.336230159
77
Post Hoc Tests (Tukey's B)
Age
Lake
n
Cavallers
Vidal d'Amunt
Veľké Hinçovo
Llebreta
Xic de Colomina
Morskie Oko
14
12
14
14
12
13
Means for groups in
homogeneous subsets
(alpha = 0.05)
1
2
4.5
5.416666667 5.416666667
5.857142857 5.857142857
6.214285714
6.25
6.384615385
Reproductive status
Lake
n
Xic de Colomina
Llebreta
Cavallers
Veľké Hinçovo
Vidal d'Amunt
Morskie Oko
12
14
14
14
12
12
Means for groups in
homogeneous subsets
(alpha = 0.05)
1
1.833333333
2.642857143
2.714285714
2.714285714
2.833333333
2.866666667
Supplementary Table ST4. Analysis of the distribution of dio2 and cyp1a mRNA
levels in male and female fish according their reproductive statusa,b
Descriptives
Sex
dio2 (log values)
Reproductive
Status
Male
Female
n
Male
Female
Std. Deviation
17
0.291158539
0.715531518
3-5
6
0.133592914
0.367284384
1-2
21
0.498250701
0.957867865
3-4
20
0.609404285
0.595431177
5-7
6
-0.16890681
0.65332076
70
0.391273746
0.758816634
1-2
17
2.935898307
0.34170676
3-5
7
3.082568227
0.482477799
1-2
21
2.76290303
0.380755823
3-4
20
2.558539516
0.472628977
5-7
6
2.476709523
0.519663266
71
2.754088168
0.452045162
Total
cyp1a (log values)
Mean
1-2
Total
ANOVA
Sum of
Squares
dio2 (log values)
Between Groups
Within Groups
Total
cyp1a (log values)
Between Groups
df
Mean Square
3.643546012
4
0.910886503
36.0868392
65
0.555182142
39.73038521
69
2.54527852
4
0.63631963
Within Groups
11.75885945
66
0.178164537
Total
14.30413797
70
Post Hoc Tests
(Tukey's B)
dio2 (log values)
Sex (Reproductive
status)
n
Female (5-7)
Male (3-5)
Male (1-2)
Female (1-2)
Female (3-4)
6
6
17
21
20
Means for groups in
homogeneous subsets
(alpha = 0.05)
1
-0.16890681
0.133592914
0.291158539
0.498250701
0.609404285
cyp1a (log values)
Sex (Reproductive
status)
n
Means for groups in
homogeneous subsets
(alpha = 0.05)
1
2
2.476709523
2.558539516
2.76290303
2.76290303
2.935898307 2.935898307
3.082568227
Female (5-7)
6
Female (3-4)
20
Female (1-2)
21
Male (1-2)
17
Male (3-5)
7
a) Defined as in supplementary Table S2
b) Reproductive status were aggregated to ensure sufficient n values for all groups
Sig.
0.174695473
0.010688093